Project Summary/Abstract Inherited BRCA1 mutations very often result in high frequency breast and ovarian cancer in affected families. This proposal is aimed at illuminating key steps that occur after menarche in tumor-free, BRCA1-mutated mammary epithelium and contribute in a major way to future BRCA1 breast cancer (BrCa) development. Our published data and work in progress suggest the existence of a series of interrelated biochemical steps that, when interrupted, result in the conversion of ostensibly normal, BRCA1- mutated breast epithelial cells into an abnormal, dedifferentiated, unusually primitive, and likely pre-malignant state. This change involves the loss of function of any member of a group of 6 particular genes which translates into a breakdown in DNA damage control which, in turn, promotes a breakdown in mammary epithelial differentiation. In breast cells these genes normally communicate with one another, functionally, as if they were components of a biologically important pathway dedicated, in part, to BrCa suppression. Indeed, their protein products form a distinct molecular complex which also contains the BRCA1 tumor suppressor protein, p220, and this complex operates in ways that suggest a major role for it in such a pathway. Among the 6 other complex members (BRG1, FancD2, CtIP,NUMB, HES-1, and BARD1), some (e.g. FancD2, BRG1) are somatically mutated in sporadic BrCa. Another (Numb) is a known BrCa suppressor in animals and a supporter of normal stem cell proliferation, and at least 5 of the 6 must be present and bind to p220 for the entire complex to form and mammary epithelial differentiation to proceed normally. Our work in progress also suggests that the integrity of this complex is required to prevent BRCA1 BrCa from developing. The proposed research will test the validity of this hypothesis along with the physiological importance of such a pathway, using a combination of cutting edge methods. Included will be an effort to a) test for lineage relationships between failed mammary epithelial cell (MEC) DNA damage repair and MEC dedifferentiation and between MEC dedifferentiation and BRCA1 tumor development and b) to decipher the mechanisms governing the interrelationships of these events. A major component of these efforts will be the application of single cell RNAseq to assess the above-noted lineage relationships and mammary epithelium- focused genomic CRISPR and protein overexpression screens to detect individual proteins that participate in these important steps in BRCA1 BrCa development. We will use a newly developed mouse model in these experiments.!